a) Field of the Invention
The invention is directed to a method and an arrangement for generating high-resolution microscopic imaging in endoscopy based on laser-induced object reaction radiation and for performing microscopic processing of biological material in endomicrosurgery.
b) Description of the Related Art
The invention is preferably applied in endoscopy based on multiphoton processes by means of femtosecond laser technology. In particular, it can be used in microimaging processes, laser scanning microscopy, optical coherence tomography, and single-photon and multiphoton imaging and is applied especially in endomicroscopy and endomicrosurgery due to the fact that the imaging or cutting optical system is miniaturized and made more flexible.
The invention is especially suitable for precision processing of biological materials such as for the optical deactivation of unwanted cells in tissue, surgical treatment of the ocular fundus and eye lens, treatment of implants, controlling delivery of active materials, jaw surgery, ear, nose and throat surgery, vascular surgery, lymph node therapy, heart surgery, neurosurgery, stem cell therapy, and tumor therapy.
Radiation from femtosecond lasers has been used hitherto predominantly in diagnostics. In particular, the two-photon fluorescence (see, e.g., U.S. Pat. No. 5,034,613 A) and SHG induced by near infrared (NIR) femtosecond laser radiation are used for three-dimensional microscopy of biological objects. Also, femtosecond lasers are used for diagnostics by means of optical coherence tomography (OCT) (WO 1998/038907).
NIR femtosecond lasers for optical cutting with a precision in the submillimeter range have been used commercially heretofore only for treatment of the cornea (e.g., EP 1 470 623 A2, DE 101 48 783 A1, U.S. Pat. No. 5,993,438 A). Multiphoton processes which lead to an ionization of the target, optical breakdown, plasma formation and to disruptive processes such as the formation and disintegration of cavity bubbles and the generation of shock waves are effective for this purpose and can be used for material cutting. By means of diffraction-limited focusing of the laser radiation by focusing optics having a high numerical aperture (NA>1) on illumination spots with a diameter of less than one micrometer, NIR laser pulses with low nanojoule pulse energy around the threshold for optical breakdown, typically in the range of 1 TW/cm2, are sufficient for carrying out material cutting. It has been demonstrated that cutting action and drill holes in the sub-200 nm range can be realized without collateral damage through the application of multiple ˜1 nJ pulses (KÖNIG et al., Optics Express 10 (2002) 171-176; KÖNIG et al., Med. Laser Appl. 20 (2005) 169-184).
Apart from applications in the field of medicine for treatment of the anterior portion of the eye, there are also femtosecond laser arrangements for precision surface machining of semiconductors and other materials (LeHarzic et al., Optics Express 13 (2005) 6651-6656).
DE 100 65 146 A1 describes a method for the analysis and optical cutting of pigmented skin tumors by means of intensive NIR femtosecond laser radiation and focusing optics having a high numerical aperture (NA). Use in the interior of the target is limited to the working distance of the macro-focusing optics with a high numerical aperture 1.2≦NA≦1.3, typically 200 μm (KÖNIG, RIEMANN, Journal Biomedical Optics 8 (3) (2003) 432-439).
Thus far, there have been no commercial light endoscopes with focusing optics having a high numerical aperture. Typical numerical apertures are in the range of low NA≈0.3. In addition, all commercial light endoscopes are based on optical materials and light guides which hinder the transmission of femtosecond pulses due to high dispersion.
There are first endoscope prototypes for use on small animals that are based on GRIN lenses and microstructured light guides with relatively low NA for two-photon image generation by means of injected fluorescence markers or prior injection of foreign DNA (transfection) which lead to the formation of fluorescing proteins. Gradient index (GRIN) lenses with typical diameters of 0.2 mm to 2 mm make it possible to construct miniaturized systems. Thanks to their planar end faces, multi-lens systems can be produced simply and compactly. The NA depends on the material that is used and on the manufacturing process of the refractive index gradient. Silver-doped GRIN lenses have a maximum NA of 0.48 (NIR at 850 nm) and thallium-doped GRIN lenses have a maximum NA of 0.55 (850 nm). However, the resolution, excitation efficiency and detection efficiency are low due to the low NA.
Extremely high laser pulse energies are required to achieve the light intensity needed for multiphoton-based removal of material which is substantially higher than the light intensity for diagnostics. This would require cost-intensive, elaborate laser apparatus. Further, there would be a high risk potential. A cutting precision in the sub-100 μm range within the target could not be achieved because of collateral damage, including the formation of large cavity bubbles in a range greater than a cell dimension and uncontrolled auto-focusing (collateral damage correlates to the pulse energy).
All of the methods and arrangements mentioned above have the disadvantage that they cannot be applied for endoscopic use of multiphoton processes by means of the radiation of a femtosecond laser inside materials and within the body for precision image generation and/or cutting with an accuracy of under one millimeter.
The development of a number of microscopic optical imaging methods (so-called microimaging) such as laser scanning microscopy (LSM), optical coherence tomography (OCT) and multiphoton imaging (MPI) has revolutionized optical microimaging and has rapidly expanded its possibilities (see, e.g., Concello et al., Nature Methods 2 (12), 2005, pages 920-931; Helmchen et al., op. cit. pages 932-940). Devices based on these methods are used increasingly in biomedicine because they permit microscopic examinations which are fast, noninvasive and do not use contrast agents (see, e.g., Flushberg et al., op. cit. pages 941-950).
At the present time, optical microscopic scanning methods are mostly used for external or near-surface examinations because flexible, miniaturized devices have not succeeded in scanning methods due to the absence of miniaturized scanners.
Existing optically imaging solutions such as, e.g., LSM endoscopes according to EP 1 468 322 have only a limited resolution and can only be used for LSM. MPI endoscopes (e.g., Ling Fu et al., Optics Express 14 (3), 2006, pages 1027-1032) intrinsically have relatively large dimensions due to the fact that a MEMS (micro-electromechanical system) mirror scanner with good optical parameters is not smaller that about 3 mm (so that an endoscope with MEMS cannot have a diameter of less than approximately 5 mm).